Bicyclic monoterpene cyclases provide the focus for study of allylic pyrophosphate cyclization, a reaction type of major importance in enzymatic C-C bond formation in the biosynthesis of numerous terpenoid natural products of pharmacological significance. A general mechanistic scheme has been proposed for the coupled isomerization-cyclization of the universal precursor geranyl pyrophosphate to various derivatives of the bornane, pinane, finchane and thujane skeletal families. The essential elements of this scheme are the ionization of the acyclic precursor, with syn-migration of the pyrophosphate moiety to afford the tertiary isomer, linalyl pyrophosphate. Following transoid to cisoid rotation, this bound intermediate cyclizes from the anti-endo conformation. The relevant model cyclases have been partially purified to remove competing activities and are being used to test the proposed scheme. 1R- and 1S-(1-3H) geranyl pyrophosphate and 3R and 3S-(1-Z-3H) linalyl pyrophosphate will be utilized as substrates, along with chromatographic resolution and stereospecific degradation of the resulting products, to delineate the overall stereochemistry of the cyclizations. The linalyl substrates will also be used to study, in isolation, the cyclization component of the reaction. Non- cyclizable substrate analogs will be employed to dissect the isomerization component. The ability of the cyclases to solvolyze sustrate analogs will be examined to define the effect of structure on binding, the ionization-isomerization step, and the partitioning of products. Specifically deuterated substrates will be utilized to examine terminating deprotonations in monoterpene olefin synthesis and to evaluate several mechanistic alternatives for cyclizations leading to complex mixtures of olefin isomers. Amino acid modifying reagents, with protection against inhibition by substrates and analogs, will be exploited to deduce the role of active site residues. FPLC techniques will be utilized to obtain homogeneous cyclases with which stoichiometric relationships will be determined and more detailed exploration of active sites initiated. The studies outlined should provide new information on the nature of these novel catalysts, allow validation of the proposed cyclization mechanism, and provide a clearer understanding of this important aspect of prenyl pyrophosphate metabolism.